Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB

Abstract

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants1,2,3,4,5,6. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive1,7,8. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes1,2,3,4. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1–4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.

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Figure 1: The Polycomb group proteins BAP1 and ASX form a conserved complex in vivo and in vitro.
Figure 2: PR-DUB is bound at Polycomb target genes in Drosophila.
Figure 3: Recombinant Drosophila and human PR-DUB deubiquitinate H2A in nucleosomes in vitro.
Figure 4: PR-DUB is required for H2A deubiquitination in Drosophila and its catalytic activity is essential for HOX gene repression.

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ArrayExpress

Data deposits

The microarray data have been deposited in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress) under the accession number E-TABM-908.

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Acknowledgements

We thank T. Sixma and G. Buchwald for the gift of proteins, H. W. Brock, R. E. Kingston, B. Korn and B. Turner for plasmids, baculoviruses and antibodies, V. Benes, J. de Graaf, S. Müller and A. Riddell for technical support, and W. Huber and J. Gagneur for discussions. T.W.M. is supported by NIH grant RC2CA148354. J.C.S., A.G.A.A., K.O., N.L.-H. and J.M. are supported by EMBL and by grants from the DFG.

Author information

Author Contributions J.C.S., A.G.A.A., K.O., N.L.-H. and J.M. conceived the project, designed and carried out the experiments, discussed and interpreted the data and prepared the manuscript. R.K.M. synthesized H2Bub1 in the laboratory of T.W.M., S.F. performed the mass spectrometry analysis in the laboratory of M.W.

Correspondence to Jürg Müller.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-8 with legends and Supplementary Table 1. (PDF 2989 kb)

Supplementary Table 2

This table shows the chromosomal coordinates of PR-DUB-bound regions and assigned target genes with gene ontology classifications. (XLS 667 kb)

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Scheuermann, J., de Ayala Alonso, A., Oktaba, K. et al. Histone H2A deubiquitinase activity of the Polycomb repressive complex PR-DUB. Nature 465, 243–247 (2010). https://doi.org/10.1038/nature08966

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